Poxviruses have long been of biomedical importance; smallpox was one of mankind's greatest plagues, and vaccination with the related vaccinia virus enabled the first eradication of a natural pathogen. In the post- vaccination era, endemic monkeypox is increasing in Africa, and the specter of the bioterrorist use of variola virus is diminished but not gone. Today, poxviruses are emerging as recombinant vaccines and promising oncolytic therapeutics. In this context, a more complete understanding of the vaccinia virus replication machinery and the breadth of virus/host interactions may uncover novel therapeutic targets and facilitate the development of more sophisticated viral recombinants for biomedical applications. Poxviruses are unique among DNA viruses that infect mammalian cells in the restriction of their infectious cycle to the cytoplasm. This autonomy from the host nucleus poses novel obstacles and offers novel opportunities for the virus. Genome replication is arguably the central process of the viral life cycle, enabling the amplification of a single infectious particle into a population of progeny genomes. During the initial phase of vaccinia virus infection, viral cores remain intact in the cytoplasm and are the sites of early gene expression, whose products include the viral DNA replication machinery. The core then undergoes disassembly and the genome is released, forming a pre-replication focus. DNA synthesis itself takes place in replication factories that are delimited by membranes derived from the endoplasmic reticulum. The core replication machinery involves the E9 polymerase, the heterodimeric processivity factor A20/UDG, the D5 AAA+ ATPase/primase, the I3 single- strand DNA binding protein (SSB), and the putative scaffold protein H5. In addition, vaccinia encodes a FEN- family nuclease, G5, that is essential for the formation of mature, complete genomes. The mechanism of genome synthesis and maturation, and of its repair after encountering intrinsic and extrinsic DNA damage, remains poorly understood. Despite the large size of the vaccinia genome, a role for cellular enzymes is likely. Our proposal will address several fundamental unanswered questions. AIM I: Life Cycle of the viral genome from core disassembly to replication initiation. This aim addresses two important questions: 1. How does core disassembly occur: what proteins are targeted for ubiquitination and how does D5 mediate disassembly? and 2. Following core disassembly, protein ubiquitination and proteasome action are prerequisites for DNA synthesis: how do these processes enable replication? AIM II: Genetic and biochemical dissection of genome replication and repair. This Aim will focus on three key questions: 1. How does the putative scaffold protein H5 contribute to replication? 2. How does the I3 SSB mediate efficient DNA replication and facilitate the maintenance viral genome integrity? 3. How does the G5 nuclease participate in genome replication and repair, and which cellular proteins contribute to maintaining the integrity of the vaccinia genome, both in the absence and presence of exogenous DNA damage?